Our laboratory takes a combination of genetic, biochemical, and visual approaches to the study of intracellular lipid trafficking in the yeast S. cerevisiae. Little is known about how the lipid composition of organelles is established and maintained or how lipids are trafficked in cells. The lipid composition of organelles is thought to be important for their proper function and for maintaining organelle identity. In addition, many diseases are associated with disregulations of intracellular lipid trafficking. 1. Sterol trafficking between the ER and plasma membrane. Cells maintain a sterol gradient across the secretory system, with the lowest concentrations in the ER and the highest in the plasma membrane (PM). Little is known about how this heterogeneous distribution of cholesterol is maintained despite continuous vesicular traffic between organelles. Both the modulation of the sterol content of transport vesicles and the non-vesicular transport of cholesterol between organelles are likely to contribute. We are investigating sterol trafficking between the ER and the PM. We have established assays for determining the rate of sterol transport between the ER and the PM in both directions. Using these assays, we have found that sterol is trafficked rapidly between the ER and PM in yeast as it is in higher eukaryotes. We then used strains with conditional defects in various proteins required for protein transport between the ER and PM and found that sterol transport was unaffected in these strains. Thus sterol trafficking does not require proteins needed for the secretory or endocytic pathways. We are now in the process of isolating mutants with defects in sterol trafficking to try to identify proteins that are directly involved in moving sterol between the ER and PM. While looking for mutants with defect in moving exogenous sterol to the ER, we have found that the MDR-like ABC transporters AUS1p and PDR11p are required. We have localized these proteins to the PM and have preliminary evidence that they are directly involved in the transport of exogenous sterol into the cell. We are in the process of further characterizing these proteins and hope to learn more about both how they transport sterol and what other proteins are required get exogenous sterol to the ER. 2. Lipid trafficking to peroxisomes, mitochondria, and lipid droplets. While both vesicular and non-vesicular lipid trafficking contribute to lipid distribution in the secretory system, lipid trafficking to peroxisomes, mitochondria, and lipid droplets probably occurs exclusively by non-vesicular mechanisms. There is no know vesicular traffic to these organelles and they do not synthesize their own lipid, thus they must receive lipid from the rest of the cell by non-vesicular pathways. Almost nothing is known about how this occurs. We have begun projects to determine how lipid is moved to these organelles. Our approach for peroxisomes and mitochondria is to target sterol and/or phospholipid modifying enzymes to these organelles in vivo. By determining the rate at which lipids are modified by these enzymes, we can estimate the rate at which lipids are being transported to the organelles. Very little is known about these rates and we have now been able to determine them. Preliminary evidence indicates that there is a large flux of both phospholipid and sterol through both peroxisomes and mitochondria despite the fact that there is no known vesicular trafficking between these organelles and the rest of the cell. With these tools in hand, we have begun to determine whether the transport of lipid to these organelles is affected in various mutants with defects in proteins that may be important for lipid movement. In addition, we have begun to isolate mutants with defects in lipid trafficking to these organelles. Finally, we have begun to establish in vitro systems to study lipid trafficking to these organelles to isolate cytosolic factors required for these processes. In a separate project we have begun looking at how lipid is transported to and from lipid droplets. These organelles are found in almost all eukaryotic cells and are important for lipid storage and homeostasis. Very little is known about how these structures form or how lipids are moved to and from them. The prevailing view in the literature is that they originate from the ER. We have found that, in yeast, lipid droplets are rather immobile organelles that tend to stay in the same part of the cell once they form and maintain a relatively constant number over time. In addition, preliminary evidence from metabolic labeling studies suggests that lipid is rapidly moved in and out of these immobile lipid particles. Therefore, soluble factors may be involved in moving lipid to and from these organelles. We are in the process of establishing an in vitro system to see if cytosolic factors are in fact required and to try to purify them. In addition, we have begun to look for mutants with defects in forming lipid particles in order to identify proteins involved in lipid particle generation.